Project Summary Pain-sensing sensory neurons of the dorsal root ganglion (DRG) and dorsal horn (DH) can become sensitized (hyperexcitable) in response to surgically induced peripheral tissue injury. Because of insufficient knowledge about the mechanisms for this sensitization, current treatment for postoperative pain has been limited to somewhat non-specific systemic drugs (opioids) having significant side effects or potential for abuse. Recent studies in our laboratory have established that CaV3.2 (T-type) calcium-channels and CaV2.3 (R-type) voltage- gated calcium channels make a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of peripheral nociceptors and controlling excitatory synaptic transmission in the DH of the spinal cord. We previously showed that the blockade of CaV3.2 currents in nociceptive DRG neurons by 5?-reduced neuroactive steroids underlies their potent peripheral anti-nociceptive effects. Our new data demonstrate that one such steroid, 3?-OH [(3?,5?,17?)-3-hydroxyandrostane-17-carbonitrile], in addition to hypnotic properties, also displays excellent spinally-mediated analgesia. We also found that 3?-OH inhibits recombinant CaV2.3 currents. This finding has led us to hypothesize that: inhibition of neuronal CaV3.2 and CaV2.3 currents in pain pathways with the novel anesthetic 3?-OH and related neuroactive steroids underlies effective analgesia. In Aim 1, we will study analgesic potency of neuroactive steroids using a clinically relevant rodent model of skin and deep tissue incision. In Aim 2, we will define the role of neuroactive steroids in modulating synaptic transmission and neuronal excitability of nociceptive DH neurons. These studies will define the whole-cell neurophysiological effects of test compounds in the major nociceptive pathway. We will also use electron microscopy to study cellular and subcellular localization of CaV2.3 and CaV3.2 channels in nociceptive DH neurons. In Aim 3, we will study mechanisms and structure-activity relationships of steroid inhibition of recombinant and native CaV2.3 currents in the nociceptive DRG neurons. The main goal of this aim is to expand the limited information currently available with the expectation that the information obtained will be important for the design of more potent and selective inhibitors of CaV2.3 channels that can be important in developing novel and safer anesthetics and analgesics. The proposed work is innovative and medically significant because we anticipate that our studies will identify novel therapies for perioperative pain that may greatly decrease the need for narcotics and potential for drug abuse.?